Reversibly 3'-O-blocked nucleotides are not only core components of reversible termination sequencing-by synthesis (SBS) but also useful building blocks for the development of template-independent enzymatic DNA and RNA synthesis strategies[1-5].
For example, 3'-O-Azidomethyl, 3'-O-Allyl and 3'-Phosphate meet the criteria for an effective blocking group[1-5]:
| Blocking group | Removal Method | Selected References |
|---|---|---|
| 3’-O-Azidomethyl | Chemical: tris(2-carboxyethyl)phosphine (TCEP) | [4] |
| 3’-O-Allyl | Chemical: Palladium-catalyzed | [4] |
| 3’-Phosphate | Enzymatic: Phosphatase | [5] |
| Reversibly 3‘-O- Blocking group | Additional Modification | (d)ATP | (d)GTP | (d)CTP | dTTP/UTP |
|---|---|---|---|---|---|
| 3‘-O-blocked dNTPs | |||||
| 3‘-O-Azidomethyl | none | 3‘-O-Azidomethyl-dATP | 3‘-O-Azidomethyl-dGTP | 3‘-O-Azidomethyl-dCTP | 3‘-O-Azidomethyl-dTTP |
| 3‘-Phosphate | none | 3‘-Phosphate-dATP | 3‘-Phosphate-dGTP | 3‘-Phosphate-dCTP | 3‘-Phosphate-dTTP |
| 3‘-O-blocked NTPs | |||||
| 3‘-O-Azidomethyl | none | 3‘-O-Azidomethyl-ATP | 3‘-O-Azidomethyl-GTP | 3‘-O-Azidomethyl-CTP | 3‘-O-Azidomethyl-UTP |
| 3‘-O-Allyl | none | 3‘-O-Allyl-ATP | 3‘-O-Allyl-GTP | 3‘-O-Allyl-CTP | 3‘-O-Allyl-UTP |
| 3‘-Phosphate | none | 3‘-Phosphate-ATP | 3‘-Phosphate-GTP | 3‘-Phosphate-CTP | 3‘-Phosphate-UTP |
| 2‘-OMe | 3‘-Phosphate-2‘-OMe-ATP | 3‘-Phosphate-2‘-OMe-GTP | 3‘-Phosphate-2‘-OMe-CTP | 3‘-Phosphate-2‘-OMe-UTP | |
Contact Barbara if you are looking for reversibly 3‘-O-blocked (d)NTPs with additional modifications!
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[1] Pichon et al. (2024) Controlled enzymatic synthesis of oligonucleotides. Commun. Chem. 7(138): https://doi.org/10.1038/s42004-024-01216-0
[2] Gao et al. (2025) Enzymatic de novo oligonucleotide synthesis: Emerging techniques and advancements. Biotechnology Advances 82:108604.
[3] Fessner (2025) Enzymatic Strategies for Next-Generation DNA Synthesis: Boosting Efficiency and Overcoming Secondary Structures. ACS Catalysis 15:16106.
[4] Wiegand et al. (2024) Template-independent enzymatic synthesis of RNA oligonucleotides. Nature Biotechnology: https://www.nature.com/articles/s41587-024-02244-w
[5] Forget et al. (2025) Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis. Nucleic Acids Research 53(4): https://doi.org/10.1093/nar/gkaf115
